Conversations with Timothy Wencewicz: Developing new antibiotics during the age of resistance

Timothy Wencewicz is an associate professor in the Department of Chemistry at Washington University in St. Louis. Established in July 2013, his lab’s main research focus is antibiotic drug discovery. A strong bias is placed on studying antibiotics isolated from nature. According to his lab page, “Much can be learned from these ‘Natural Products’ whose molecular scaffolds evolved over billions of years to solve many of the puzzles behind effective antibiotics.” He discussed some of his research with us.

SCIENTIFIC INQUIRER: What is a beta-lactam ring? What is its structure and mechanism? Where is it found?

TIMOTHY WENCEWICZ: The beta-lactam and beta-lactone are two members of related ring sets better known as strained heterocycles. They carry a large amount of strained energy which makes them very reactive. The classic beta-lactams — which have a nitrogen in the ring — include molecules like penicillin. They are basically like mini-molecular bombs that go off inside an enzyme active site. Beta-lactams work by interacting with an enzyme called a transpeptidase that cross-links bacterial cell walls to make it stronger. When you inhibit that enzyme, it turns the normally rigid cell into something like a spaghetti string and it bursts open. That is because of the strained beta-lactam ring and is basically the universal mechanism for our most successful antibiotics.

SI: And what is a beta-lactone?

TW: The cousin to the beta-lactam is the beta-lactone. That is the ring that my lab studies. Far less is known about that ring. It has found clinical usefulness because it carries the same reactivity as a lactam. It’s a four member ring and has an oxygen instead of a nitrogen. That small change makes the reactivity the same but changes the type of enzymes it likes to inhibit. They tend to not inhibit the transpeptidases that cross link bacterial cell walls. Instead, they inhibit enzymes related somewhat chemically known as lipases or hydrolases. The classic example of a beta-lactone used in humans is the molecule orlistat/xenical. It’s a human lipase inhibitor. Its primary mode of action is the same. It mimics the fatty acid and binds to the lipase. Instead, the lipase encounters this very reactive strained four-member ring. It explodes inside the enzyme active site forming a covalent adduct which is the most powerful of adduct because it is a permanent covalent bond.

SI: Can you discuss how you started working with beta-lactones?

TW: The beta-lactam has been around for a long time. They were developed during wartime to save soldiers on the battlefield. Their widespread distribution was enabled by a knowledge of how nature produces these molecules. Penicillin comes from the penicillium mold. The strain used to overproduce penicillin came from a cantaloupe. That’s the primary way we get molecules like penicillins – through fermentation of the microbes that produce them.

Today we can go one step further. We can sequence their genomes and reveal the code that allows the microbe to produce the molecule. That allows us to have a deeper understanding of the chemistry behind how the pieces are put together and allows us to look for a wider range of related molecules that might be the next penicillin. When we started our work with beta-lactones, there was no known enzyme known for making the beta-lactone ring. We basically went enzyme hunting.

We chose a molecule for our studies called oblaflorin. We sequenced the entire genome of pseudomonas fluorescens, a bacterium associated with plant roots. It’s a symbiont. We were able to locate the DNA sequence responsible for producing obaflorin. Right away we saw something special in the gene cluster. It was an unusual enzyme and there was a thioesterase domain sitting at the end of a non-ribosomal peptide synthetase (NPRS). It’s a fascinating class of enzymes because they represent an alternative way of making peptides, independent of the ribosome.

The advantage of the NRPS in making molecules like penicillin is that you are not limited to the canonical, small selection of proteinogenic amino acids that make up our common everyday proteins. An NRPS can take any amino acid substrate it wants. There are thousands of amino acid substrates that never make it onto a protein on a ribosome but they make it onto peptides through NRPS. The domain we found on our NRPS kicks the molecule off by cyclizing into the beta-lactone ring. It forms at the very last step in a very elegant manner and completes the biosynthesis of the molecule. It is highly efficient.

We were perplexed how it works because making a strange ring seems to require energy because they are high energy species. This enzyme cleverly solves the problem by making key mutations in the thioesterase domain. The most critical is the active site residue. A serine is mutated to a cysteine which has a thiol functional group. The thioester intermediate is much higher in energy. This enables the formation of the high energy ring. We revealed the biochemical blueprint of how nature forms beta-lactone rings which allows us to search all publicly deposited DNA sequences to see what other kinds of bacteria or fungi can make this molecules.

SI: You mentioned how elegant your enzymatic platform is. Do you foresee ways of refining the process further?

TW: Absolutely. NRPS are very flexible in the type of amino acids they can take in. They also have gatekeepers since they can’t just take any amino acid. Selectivity of what amino acids go into the assembly line is an issue. We’ve been exploring it. We’re pushing the limits of what the enzyme can do. We’re going to see how far we can push substrate preference.

SI: Where do the beta-hydroxy amino acids come from?

TW: We put the entire pathway together. You’ll often see a paper with one enzyme or two. We characterized all five in a row. We did the key transformations from very basic building blocks into more exotic high value products commonly associated with secondary metabolism, metabolism not essential for life but offers some advantage.

SI: What bacteria does oblaflorin work best against?

TW: Oblaflorin is a broad spectrum antibacterial agent. It has a bacteriostatic effect which means it does not kill bacteria outright but it halts them from growing. The original isolation papers were by Squibb Institute, now Bristol Myers. They showed that oblaflorin was effective in a mouse model of strep infection. It’s a sepsis model where they basically infect the blood of the mouse with lethal doses of strep and then they give it the compound and see if they can save its life.

It was effective.when we were choosing oblaforlin that there weren’t many papers. There were about five. That was striking to us because when we first isolated oblaflorin we noticed that the moment you drop it into water it hydrolyzes. It decomposes. We thought how can this be an effective antibiotic if it’s so unstable. We realized that if it’s so unstable in water the only way to keep enough compound around to be effective is to have a fast enzyme making it. We believe this compound, though unstable, reacts quickly with its biological target. We don’t know what that is yet.

In the case of the plant bacterial interaction, pseudomonas fluorescens makes a biofilm on the plant roots and they can continuously crank out oblaflorin all the time with the enzyme system.

SI: Bacteria that are resistant to a certain class of antibacterial compounds tend to be predisposed to other antibiotics of that class. Does that hold the same for beta-lactones as cousins of lactams?

TW: Yes. So beta-lactones are also susceptible to the same resistance mechanisms that work against the beta-lactams. Those are beta-lactamase enzymes. That is how the Squibb Institute discovered oblaflorin. They were using a strain of E. coli that produced a beta-lactamase and a twin brother that did not produce it. They were looking for compounds like beta-lactams where they worked effectively against E. coli that did not express beta-lactamases but lost their activity against the e coli that expressed the beta lactamases. That’s where their serendipitous discovery of beta-lactones happened. They realized that they were susceptible to the same resistance mechanisms.

With all resistance mechanisms, it’s a question of timing. The beta-lactamases work so effectively on beta-lactams because they are almost perfect enzymes. What do I mean by a perfect enzyme? It makes no mistakes. Meaning every time a beta-lactamase collides with penicillin it reacts with it. It makes no mistakes. Its a diffusion controlled reaction. The rate of the reaction is limited by how quickly the enzyme and the molecule can diffuse together and collide. That’s why the beta-lactamases are so good. They are so fast. Will they be as fast against the beta-lactones? We don’t know. We still don’t know the target of oblaflorin. That’s the challenge with discovering new antibiotics. Once you find the compound it takes a long time to work out the details.

You’ll hear people say we are in an antibiotic crisis, we have no molecules. We are in a crisis but it’s not true that we have no molecules. We have lots. It just takes a lot of time and money to study each individually to see whether they are effective in humans.

SI: You mention the challenges of drug discovery. Considering those challenges, will we enter a post-antibiotic era?

TW: It’s possible. There are some that argue we are already there. There are infections that are resistant to every FDA approved antibiotics. These are pathogens that we could kill in 1950. That is definitely a step back. But bacterial infections are very large global killers but the really bad superbugs, the multidrug resistant pathogens, are still infecting and killing a small percentage of people when you think about it in large numbers. The worst is MRSA which accounts for about 100,000 infections in the US and 19,000 deaths. That’s a lot. MRSA can be treated though. There are still some last-resort antibiotics.

You can look at certain examples of pathogens like tuberculosis that infects 1 in 3 people in the world. Now multidrug resistant strains of it are popping up. We can still treat it but it’s getting harder. I don’t think we’ve jumped off the cliff yet, but there has to be a change in how we approach the development of antibiotics. There is one thing that it comes down to. Money. There’s not much market in a cure.

SI: How did you choose a life in science? Did you always want to be a scientist?

TW: I don’t think I’ve always wanted to be a scientist. I think I was like everybody in rural USA. I probably wanted to be a baseball player. I was lucky. I had great teachers and supportive parents. My father was a mathematics professor for forty-three years at Southeast Missouri State University. I used to really enjoy hanging around his faculty office area, drawing on the chalkboard. I started off as a math major but chemistry really spoke to me. I’m a more qualitative person at heart. I like mixing drops together and seeing what happens – the most fundamental thing about chemistry. In graduate school, I saw that chemistry makes such a big difference in the world and that it drives things like the pharmaceutical industry. I knew when I went to University of Notre Dame that I wanted to work on pharmaceuticals and specifically antibiotics. I think the global call to service for antibiotics is great. If you go into antibiotics you aren’t going in for the money. You’re going to save people’s lives. I know that the discoveries I’m making will be part of the public domain and collective wisdom of science and someday someone else might look at it and think we can do something with that.